Method of making biofilms

ABSTRACT

Blofilm forming organisms are incubated to form a biofilm on projections by providing a flow of liquid growth medium across projections, the direction of the flow of liquid being repeatedly changed, and an assay made of the resulting biofilm. Biofilm forming organisms are incubated to form a biofilm on projections arranged in rows, with several projections in each row, while providing a flow of liquid growth medium across each row of projections, and an assay made of the resulting biofilm. Sensitivity of the biofilm to antimicrobial reagent may be determined by treating the projections with antimicrobial reagent before carrying out the assay, by treating each row of projections with a different antimicrobial reagent, and each of the projections in a row with a different concentration of antibacterial reagent. A biofilm assay device includes a vessel including at least one channel for flow of liquid growth medium, projections arranged in at least one row and having a support for supporting the projections within the channel, and a tilt table to flow liquid growth medium along each channel in different directions across the projections. A further biofilm assay device includes a vessel including channels for flow of liquid growth medium, projections arranged in rows and having a support for supporting the projections within the channels, and a rocking table to rock the vessel and flow liquid growth medium along each channel across the projections.

FIELD OF THE INVENTION

[0001] This invention relates-to methods and apparatus for theincubation and analysis ofiiofilms, and analysis of sensitivity ofbiofilms to antimicrobial reagents.

BACKGROUND OF THE INVENTION

[0002] Extensive study into the growth properties of bacteria in recentyears has shown that they form complex layers that adhere to surfaces.These complex forms of bacteria are known as biofilms, or sessilebacteria. Biofilms may cause problems in a variety of areas includingthe bodies of humans and animals, food processing, health carefacilities, metal-working shops, dairy farms and other industries.

[0003] It is now widely known that bacteria in the form of biofilms aremore resistant to antimicrobial reagents than planktonic bacteria. Yettesting for the presence of bacteria and the testing of the efficacy ofantibiotics against bacteria has traditionally involved testing forplanktonic bacteria. Thus, bacterial inhibitory concentration of theantimicrobial reagent may be underestimated, with the result that thewrong antimicrobial reagent or wrong amount of antimicrobial reagent maybe used for the treatment of the bacteria.

[0004] One type of device for monitoring biofilm buildup is described inthe Canadian Journal of Microbiology (1981), volume 27, pages 910 to917, in which McCoy et al describe the use of a so-called Robbins devicewhich comprises a tube through which water in a recycling circuit canflow. The tube has a plurality of ports in its walls, each beingprovided with a stud having a biofoulable surface and being capable ofbeing retained in the port in fixed relationship with respect to thetube so that the biofoulable surface forms part of the internal surfaceof the tube. The studs may be-removed from the ports after a desiredtime interval and the test surfaces by microscopy of the surfacesanalyzed for growth of microorganisms or by removal of themicroorganisms from the surfaces and subsequent estimation of the degreeof growth. The number of microorganisms can be estimated for instance byphysical or chemical means, e.g. by detection of bacterial ATP or byfurther culturing the microorganisms and analyzing the products.

[0005] In another device described in U.S. Pat. No. 5,349,874, biofilmgrowth in a water carrying conduit is determined by providing pluralremovable studs in the conduit or in a second conduit parallel to thefirst. The studs may be removed for analysis of biofilm on the studs.Such devices as the Robbins device, or others using removable studs in asingle conduit, result in rather lengthy processing times, and do notprovide rapid response times for the testing of several differentantimicrobial reagents.

[0006] In a still further device, described in “Simple Method forMeasuring the Antibiotic Concentration Required to Kill AdherentBacteria”, Miyake et al, Chemotherapy 1992; 38, 286-290, staphylococcusaureus cells adhered to the bottom of a 96 well plastic tissue cultureplate were treated with serially diluted antibiotic solutions, andviability of the cells was judged by their growth after a further 24hours incubation. This method has the disadvantage of inconsistentcolonization of sessile bacteria and settling of planktonic bacteria.

[0007] The device described in this patent document allows for artefficient and automated biofilm killing assay that has particular usewith the 96 well platform common to many diagnostic assay systems.

SUMMARY OF THE INVENTION

[0008] There is therefore provided in accordance with one aspect of theinvention, a method of growing a biofilm, in which biofilm formingorganisms are incubated to form a biofilm on plural biofilm adherentsites by providing a flow of liquid growth medium across the pluralbiofilm adherent sites, the direction of the flow of liquid beingrepeatedly changed.

[0009] In a further aspect of the invention, biofilm forming organismsare incubated to form a biofilm on plural biofilm adherent sitesarranged in plural rows, with plural biofilm adherent sites in each row,while providing a flow of liquid growth medium across the plural biofilmadherent sites.

[0010] In a further aspect of the invention, an assay is made of acharacteristic of the resulting biofilms.

[0011] In a further aspect of the invention, the characteristic of thebiofilm is the sensitivity of the biofilm to antimicrobial reagent andthe method further includes, before assaying the biofilm, treating thebiofilm adherent sites with antimicrobial reagent.

[0012] In a further aspect of the invention, there is also included thestep of, after treating the biofilm adherent sites with antimicrobialreagent, dislodging the biofilm from the biofilm adherent sites andfurther incubating the biofilm. Dislodging the biofilm from the biofilmadherent sites may include dislodging the biofilm from each biofilmadherent site into a separate well of a microtiter plate.

[0013] When the biofilm adherent sites are formed in rows, treating thebiofilm adherent sites with an antimicrobial reagent may includetreating each row of biofilm adherent sites with a differentantimicrobial reagent, and treating each of the biofilm adherent sitesin a row with a different concentration of antimicrobial reagent.

[0014] In a further aspect of the method of the invention, differentbiofilm adherent sites are treated with different antimicrobialreagents, such as different combinations of antimicrobial reagents asmight be used in testing the efficacy of various modifications of asingle antimicrobial reagent.

[0015] Preferably, the flow direction of the liquid growth medium isrepeatedly reversed. In this aspect of the invention, the liquid growthmedium may flow in channels of a vessel, and the direction of flow ofthe liquid growth medium is reversed by rocking of the vessel.

[0016] In a further aspect of the invention, the biofilm adherent sitesare projections from a lid and incubating the biofilm includessuspending the projections in liquid growth medium in the channels whilerocking the vessel so as to provide shear forces on the biofilm duringgrowth of the biofilm.

[0017] There is further provided, in accordance with an aspect of theinvention, apparatus for analyzing biofilms, the apparatus comprising avessel including at least one channel for flow of liquid growth medium.Plural biofilm adherent sites are arranged in at least one row and havea support for supporting the plural biofilm adherent sites within thechannel.

[0018] In accordance with a further aspect of the apparatus according tothe invention, there is provided means to flow liquid growth mediumalong each channel, preferably in different directions, across theplural biofilm adherent sites.

[0019] In accordance with a still further aspect of the apparatus of theinvention, there is provided means to avoid contamination of the insideof the vessel.

[0020] Preferably, the plural biofilm adherent sites are formed inplural rows, with plural sites in each row, and the vessel includesplural channels, with one channel for each row of plural biofilmadherent sites.

[0021] In a further aspect of the invention, the support for the pluralbiofilm adherent sites forms a lid for the vessel.

[0022] In a still further aspect of the invention, the means to flowliquid growth medium across the plural biofilm adherent sites is a tilttable.

[0023] These and other aspects of the invention will be made apparent inthe description and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] There will now be described preferred embodiments of theinvention, with reference to the drawings, by way of illustration, inwhich like numerals denote like elements and in which:

[0025]FIG. 1 is a bottom view of plural biofilm adherent sites on a lidof a vessel;

[0026]FIG. 2 is a top view of a vessel for receiving the plural biofilmadherent sites of FIG. 1;

[0027]FIG. 3 is a side view, partly broken away, of the lid and vesselof FIGS. 1 and 2;

[0028]FIG. 4 is a side view schematic of a lid and vessel combination asshown in FIG. 3 on a tilt table;

[0029]FIG. 5 is a top view of a 96 well plate for use with theinvention; and

[0030]FIG. 6 is a side section of hollow projection for use with theinvention showing how it may be sealed.

DETAILED DESCRIPTION OF PREFERREDEMBODIMENTS/BEST MODE FOR CARRYING OUTTHE INVENTION

[0031] As shown most particularly in FIGS. 1, 2 and 3, the biofilm assaydevice includes a biofilm lid 10 composed of ELISA grade plastic orother suitable material (eg. stainless steel, titanium) with projections12 from the lid 10. The projections 12 form biofilm adherent sites towhich a biofilm may adhere. The projections 12 are preferably formed inat least eight rows 14 of at least twelve projections each. Othernumbers of rows or numbers of projections in a row may be used, but thisis a convenient number since it matches the 96 well plates commonly usedin biomedical devices. Additional projections as shown may be used todetermine the initial-biofilm concentration after incubation. Theexemplary projections 12 shown are about 1.5 cm long and 2 mm wide.

[0032] The biofilm assay device also includes a vessel 20 which includesa liquid holding basin 22 divided into plural channels (troughs) 24 bymoulded ridges 26. The channels 24 are wide enough to receive theprojections 12. There should be one channel 24 for each projection 12 ofany given row 14. The lid 10 forms a support for the projections 12 forsupporting the biofilm adherent sites within the channels 24. The lid 10has a surrounding lip 16 that fits tightly over a surrounding wall 28 ofthe vessel 20 to avoid contamination of the inside of the vessel duringincubation.

[0033] If existing devices are used to carry out the invention, thenthey should be modified for use with the invention. In the case of theFalcon system, wherein projections are fitted in removable strips, thestrips should be sealed, for example by using a plastic sheet placedover the strips. In the Falcon system, there is also a trough barrierthat extends-across the entire vessel. This isolates one channel, and ifthe Falcon device is to be used, should be removed or modified toterminate like one of the ridges 26.

[0034] If the NUNC system is used, which uses hollow projections, as forexample shown in FIG. 6, a sheet should also be used to cover theprojections in the case when they are broken off. Thus, as shown in FIG.6, the projection 12 a is covered with a sheet 13 to preventcontamination of the inside of the vessel. The NUNC device also requiresmodification of the connection of the lid to the vessel in order toavoid contamination of the inside of the vessel, such as by raising aninternal barrier to seal against the lid.

[0035] The biofilm incubation vessel 20 serves two important functionsfor biofilm development. The first is as a reservoir for liquid growthmedium containing the biofilm forming organisms which will form abiofilm on the projections 12 of the biofilm lid 10. The second functionis to generate shear force across the projections, which allows foroptimal biofilm production on the projections. The biofilm formingorganisms may be for example, bacteria, yeast and fungi, such asfilamentous fungi.

[0036] As shown, in FIG. 4, shear force on the projections 12 isgenerated by rocking the vessel 20 with lid 10 on a tilt table 30. Theprojections 12 sit suspended in the channels 24, so that the tips of theprojections 12 may be immersed in liquid growth medium flowing in thechannels 24. The ridges 26 channel the liquid growth medium along thechannels 24 past and across the projections 12, and thus generate ashear force across the projections. Rocking of the vessel 10 causes arepeated change in direction of flow, in this case a repeated reversalof flow of liquid growth medium, across the projections 10, which helpsto ensure a biofilm of equal proportion on each of the projections 12 ofthe lid 10. While it is possible to grow biofilm with only one directionof flow of liquid growth medium, the use of an array of projections 12suspended in several channels makes the arrangement difficult toconstruct, since then some method would need to be found to recirculatefluid around from one end of each channel to the other end. Rocking ofthe vessel, with liquid flowing backwards and forwards along thechannels, provides an excellent biofilm growth environment thatsimulates natural occurring conditions of turbulent flow.

[0037] Each projection 12 and each channel 24 should have substantiallythe same shape (within manufacturing tolerances) to ensure uniformity ofshear flow across the projections during biofilm formation. In addition,the uniform channels 24 should all be connected so that they share thesame liquid nutrient and mixture filling the basin 22. With sharing ofthe same biofilm forming soup and channel configuration being the samefor each channel, biofilms are produced at each projection that areequivalent for the purpose of testing antimicrobial reagents. In thisway, different concentrations of different antibiotics may be comparedto each other without regard to positional variance of the projections.Biofilms thus produced are considered to be uniform. Results have beenobtained within P<0.05 for random projections on the plate.

[0038] Sensitivity of a biofilm to antibiotic or biocide, referred to inthis patent document collectively as “antimicrobial reagent”, ismeasured by treating the biofilm adherent sites with an antimicrobialreagent, and then assaying the biofilm. This may be accomplished byplacing the lid 10, which was colonized with a bacterial biofilm in anincubation vessel 12, into a conventional 96 well plate 40 such asillustrated in FIG. 5, the number of wells 42 being determined by thenumber of projections 12, in which growth medium containing antibioticor biocide dilutions has been dispensed. The lid 10 and plate 40 fitsuch that bacterial contamination from outside the plate cannot takeplace. Projections 12 that have been incubated in the same channel 24 ofthe vessel 20 should each be treated with a different antimicrobialreagent. In this manner, consistent results may be obtained since thegrowth conditions in any one channel will be very similar along theentire channel and thus for each projection 12 suspended in thatchannel. This helps improve the reliability of treatment of differentprojections 12 with different antimicrobial reagents.

[0039] In addition, for the step of incubating the biofilm aftertreatment with an antimicrobial reagent, a conventional cover (notshown) made of the same plastic as the 96 well plate 40 is required soas to prevent contamination of the 96 well plates 40 during incubationand to form a very low profile with the plate 40 as to make the coveredplate accessible to standard ELISA plate readers. For each assay, two 96well plates 40 and plate covers will be needed to provide thetraditional Minimum Inhibitory Concentration (MIC) and the MinimumBiofilm Eliminating Concentration (MBEC).

[0040] Procedure

[0041] For each organism a biofilm growth curve should be determined toensure the biofilm has reached satisfactory proportion to be tested forantibiotic/biocide sensitivity.

[0042] Day 1—incubate bacteria of choice in a suitable growth medium,which may be tryptic soy broth (TSB), overnight with shaking at asuitbahe incubation temperature under optimal oxygen tension. Thetemperature may be, in each case of incubation described in this patentdisclosure, 37° C.

[0043] Day 2—Fill the vessel 20 of the biofilm assay device system with20 ml of a 2% overnight culture suspended in fresh growth medium (egTSB); cover with projection covered lid 10 and incubate on a rockingplatform (Bellco Rocker Platform), for example at setting 3 in a 37° C.incubator. Aseptically remove 2 projections per hour starting at hour 2of the growth curve and continuing for 7 hours. Each projection isrinsed by gentle dipping in sterile phosphate buffered saline (PBS), andthen placed in 1 ml of sterile phosphate buffered saline and sonicatedfor 5 min. using a DoALL Ultrasonic Cleaner at 21 kc to dislodge thebiofilm. The sonicate is serially diluted (10⁻¹ through 10⁻⁶) inphosphate buffered saline and spot plated to determine biofilm count.

[0044] Day 3—Plates are counted to determine the rate of colonizationand to calculate the incubation time required to achieve a biofilm of˜10⁵ colony forming units (cfu)/ projection, which is an optimal biofilmthickness for measuring antibiotic/ biocide killing.

[0045] MBEC Assay

[0046] Day 1—Inoculate growth medium (eg TSB) with bacteria and growovernight, for example at 37° C. with shaking.

[0047] Day 2: (1) Place 20 ml of a 2% overnight culture diluted in freshgrowth medium (for example TSB) in the vessel 20 of the biofilm assaydevice system, cover with the biofilm lid 10 and incubate on the rockingplatform 30, for example at 37° C. for the time required to reach abiofilm of ˜10⁵ cfu/projection 12. Change of direction of flow of liquidgrowth medium caused by the rocking of the tilt table 30 enhances thegrowth of a biofilm.

[0048] (2) While the biofilm is incubating, in a sterile hood prepareantibiotic stock solutions at 5 mg/ml in sterile double distilled water(ddw) or biocide as per directions of the producer, and prepareantibiotic/biocide dilutions in growth medium (for example TSB) in a 96well plate. Up to 8 different antibiotics or biocides can be tested byusing 1 row (A-H) for each antimicrobial reagent. Plates are set up asfollows:

[0049] Rows A-H each contain a specific antibiotic/biocide. Wells ineach row are set up with different concentrations of antimicrobialreagent as follows:

[0050] Well #1 TSB no-antibiotic

[0051] Well #2 TSB plus 1 mg/ml antibiotic

[0052] Well #3 TSB plus 2⁻² mg/ml antibiotic

[0053] Well #4 TSB plus 2⁻³ mg/ml antibiotic

[0054] Well #5 TSB plus 2⁻⁴ mg/ml antibiotic

[0055] Well #6 TSB plus 2⁻⁵ mg/ml antibiotic

[0056] Well #7 TSB plus 2⁻⁶ mg/ml antibiotic

[0057] Well #8 TSB plus 2⁻⁷ mg/ml antibiotic

[0058] Well #9 TSB plus 2⁻⁸ mg/ml antibiotic

[0059] Well #10 TSB plus 2⁻⁹ mg/ml antibiotic

[0060] Well #11 TSB plus 2⁻¹⁰ mg/ml antibiotic

[0061] Well #12 TSB plus 2⁻¹¹ mg/ml antibiotic

[0062] (3) After the incubation period, break off 2 projections 12 (froman area of lid 10 that will not be used in the antibiotic assay, i.e. anunused row) rinse and sonicate in PBS, dilute and spot on plates todetermine the initial biofilm concentration (exactly as done on thegrowth curve). Rinse the remaining projections in PBS using a troughedvessel 20 and place the colonized projections into the 96 wellantibiotic plate and incubate overnight at 37° C.

[0063] Day 3: Depending on thetechnology used to assay the incubated andtreated biofilm, the biofilm may be assayed in place, withoutdislodging, or may be dislodged from the projections and then assayed,or may be dislodged, further incubated and then assayed. The followingsteps show an example in which the biofilm is dislodged, incubated andassayed.

[0064] (1) In a sterile hood remove biofilm lid 10 from the 96 wellantibiotic plate (do not discard the plate), rinse the projections in a96 well plate containing sterile PBS, then place the lid 10 in a fresh96 well plate containing fresh TSB (0.25 ml per well) and sonicate theentire biofilm lid 10 to dislodge the surviving biofilm bacteria, withthe biofilm from any one projection 12 being dislodged into a separatewell of the 96 well plate. Again in the hood remove the biofilm lid 10and replace with a flat cover.

[0065] (2) Read the antibiotic plate at 490 nm for indication ofplanktonic survival, or use some other method, such as are known in theart, for assaying planktonic survival.

[0066] (3) From the freshly sonicated plate remove 0.02 ml of the mediumfrom each well and spot plate neat; and 0.025 ml from each well toprepare a serial dilution (10⁻¹ to 10⁻⁶) and spot plate 0.02 ml(dilutions can be done in microtiter plates, one for each antibiotic).Place the plate containing the remaining medium and bacteria to incubateat 37° C., overnight.

[0067] Day 4: Assay the sessile survival, by for example reading theplate incubated overnight at 490 nm or by counting the colonies on theplates to determine the actual number of surviving bacteria, or by usingsome other method known in the art.

[0068] Several different coentional methods may be used to count thebacteria. It may be done by incubating the sonicated bacteria, takingserial dilutions and visually counting the colony forming units, orautomated methods may be used, as for example using an optical reader todetermine optical density. It has been found however that the opticalreader of turbidity is too imprecise for practical application, and itis preferred that vital dye technology be applied to automate themeasurement of viability, by treating the biofilm with a vital dye, andmeasuring the intensity of light given off by the dyed biofilm. In thecase of using vital dye technology, the biofilm need not be furtherincubated. In a further embodiment, the assay may be carried out bysonicating the cells until they lyse and release ATP and then addingluciferase to produce a mechanically readable light output. In a stillfurther embodiment, the assay may be carried out directly on the biofilmon the projections using a confocal microscope, although it should beconsidered that this is difficult to automate. In the examples thatfollow, the results are obtained from a manual count following serialdilution. Examples are given to show a comparison between counts ofplanktonic bacteria and counts of sessile bacteria for the same growthconditions.

Example #1

[0069] Table 1 shows the results of incubation of staphylococcusepidermidis biofilm on projections 12 in channels 24 while rocking thevessel 20, followed by treatment with antibiotics A (Ancef, cefazolin),B (Orbenin-2000, cloxacillin), C (Primaxin, imipenem and cilastatin), D(Vancomycin), E (Dalacin, clindamycin) and F (Tazadine, ceftazidine) indoubling dilutions. The antibiotic was applied to the wells 2-12 in rowsA-F of a 96 well plate as follows: well 2: no antibiotic, well 3: 1000μg/mL, well 4: 500 μg/L . . . well 12: 2 μg/mL. Results are given inTable 1 terms of cfu/projection from a manual reading. TABLE 1 2 3 4 5 67 8 9 10 11 12 A 5.5 × 1.1 × 4.5 × 4.0 × 4.5 × 1.2 × 6.0 × 5.5 × 3.0 ×1.4 × 2.1 × 10⁴ 10⁴ 10³ 10³ 10³ 10⁴ 10⁴ 10⁴ 10⁵ 10⁵ 10⁶ B 1.7 × 6.0 ×1.1 × 1.2 × 2.0 × 2.0 × 2.2 × 1.6 × 1.6 × 2.6 × 9.5 × 10⁴ 10² 10³ 10³10³ 10³ 10⁴ 10³ 10⁴ 10⁴ 10⁴ C 9.0 × 8.5 × 1.2 × 1.9 × 3.2 × 6.5 × 5.5 ×6.5 × 1.0 × 1.5 × 1.1 × 10³ 10² 10³ 10³ 10⁴ 10⁴ 10⁴ 10⁴ 10⁵ 10⁵ 10⁶ D1.2 × 1.1 × 7.0 × 1.3 × 1.9 × 6.5 × 8.0 × 2.5 × 2.5 × 5.0 × 2.5 × 10⁴10³ 10² 10³ 10³ 10³ 10³ 10³ 10³ 10³ 10⁵ E 1.0 × 3.5 × 6.5 × 5.0 × 1.2 ×1.0 × 7.5 × 8.0 × 2.5 × 8.5 × 1.9 × 10⁴ 10³ 10³ 10³ 10⁴ 10⁴ 10⁴ 10⁴ 10⁴10⁴ 10⁶ F 5.5 × 1.9 × 2.3 × 4.5 × 3.5 × 1.4 × 1.9 × 2.5 × 4.0 × 3.5 ×2.7 × 10⁴ 10³ 10³ 10³ 10⁴ 10⁴ 10⁵ 10⁴ 10⁴ 10⁴ 10⁵

[0070] Table 2 gives the optical density readings of turbidity of thesame staphylococcus epidermidis biofilm, treated in the same manner asthe samples of Table 1, to show a comparison between the manuallycounted cfu and the automated reading. TABLE 2 2 3 4 5 6 7 8 9 10 11 12A .951 .856 .971 1.010 .961 .984 .993 1.036 .996 .967 .980 B .900 .935.890 .915 .902 .998 .944 .919 .909 .963 .938 C .914 .843 .768 .792 .870.907 .869 .927 .863 .908 .959 D .898 .524 .708 .805 .884 .898 .854 .835.901 .907 .958 E .901 .869 .894 .851 .858 .936 .873 .907 .874 .937 .938F .905 .897 .888 1.013 .855 .992 .903 .920 .850 .843 .902

[0071] Table 3 shows the optical density readings of turbidity ofstaphylococcus epidermidis planktonic bacteria incubated and treatedwith antimicrobial reagent under the same conditions as for the resultsshown in Table 1. This table clearly shows how the assay of planktonicbacteria gives a lower count in many instances than the assay of sessilebacteria incubated and treated under the same conditions. TABLE 3 2 3 45 6 7 8 9 10 11 12 A .959 .353 .381 .411 .403 .409 .413 .411 .599 .574.610 B .927 .346 .375 .400 .404 .442 .422 .408 .430 .562 .585 C .950.409 .411 .431 .426 .447 .475 .476 .504 .553 .620 D .907 .351 .374 .403.410 .409 .421 .404 .415 .413 .406 E .903 .861 .845 .907 .913 .887 .959.878 .910 .890 .929 F .905 .359 .383 .397 .410 .466 .540 .615 .627 .694.853

Example #2

[0072] Table 4 shows the results of incubation of staphylococcus aureusbiofilm on projections 12 in channels 24 while rocking the vessel 20,followed by treatment with antibiotics A (Ancef, cefazolin), B(Orbenin-2000, cloxacillin), C (Primaxin, imipenam and cilastatin), D(Vancomycin), E (Dalacin, clindamycin) F (Tazadine, ceftazidine) and G(Ciprofloxacin) in doubling dilutions. The antibiotic was applied to thewells 2-12 of the rows A-G of a 96 well plate as follows: well 2: noantibiotic, well 3: 1000 μg/mL,. well 4: 500 μg/mL . . . well 12: 2μg/mL. TABLE 4 2 3 4 5 6 7 8 9 10 11 12 A 2.0 × 10⁵ 0 0 0 50 50 50 × 10²50 5.0 × 10² 1.0 × 10⁴ 2.0 × 10⁴ B 5.0 × 10⁵ 0 50 0 0 50 1.0 × 10³ 0 4.5× 10² 50 9.0 × 10² C 1.5 × 10⁵ 0 0 0 0 0 0 0 0 0 1.0 × 10² D 6.0 × 10⁵ 00 0 0 0 0 0 0 0 1.5 ×10⁴ E 4.5 × 10⁵ 0 0 0 0 0 5.0 × 10² 50 0 0 2.0 ×10³ F 2.0 × 10⁵ 0 1.0 × 10² 0 1.0 × 10² 50 0 0 1.0 × 10⁵ 4.0 × 10⁵ 9.0 ×10⁵ G 3.5 × 10⁵ 0 0 0 50 1.0 × 10² 50 0 0 0 0

[0073] Table 5 shows the-optical density readings of turbidity of thesame staph. aureus biofilm, treated in the same manner as the samples ofTable 4, to show a comparison between the manually counted cfu and theautomated reading. TABLE 5 2 3 4 5 6 7 8 9 10 11 12 A .963 .333 .414.461 .758 .666 .647 .649 .673 .845 .853 B .924 .073 .626 .072 .791 .771.832 .071 .803 .858 .838 C .913 .073 .073 .071 .073 .073 .071 .072 .071.617 .822 D .903 .073 .073 .071 .093 .071 .071 .070 .071 .073 .903 E.942 .073 .074 .070 .071 .072 .487 .627 .069 .096 .867 F .938 .779 .830.777 .739 .719 .840 .806 .882 .924 .916 G .929 .073 .072 .073 .778 .822.793 .121 .071 .072 .068

[0074] Table 6 shows the optical density readings of turbidity of staph.aureus planktonic bacteria incubated and treated with antimicrobialreagent under the same conditions as for the results shown in Table 4.This table clearly shows how the assay of planktonic bacteria gives alower count in many instances than the assay of sessile bacteriaincubated and treated under the same conditions. TABLE 2 3 4 5 6 7 8 910 11 12 A .935 .064 .061 .065 .067 .066 .067 .068 .063 .058 .057 B .691.070 .071 .077 .071 .069 .069 .073 .068 .067 .059 C .880 .116 .106 .097.087 .081 .078 .078 .081 .074 .072 D .891 .071 .074 .081 .077 .075 .078.078 .076 .075 .075 E .857 .069 .074 .080 .077 .080 .075 .078 .076 .084.264 F .895 .073 .076 .081 .079 .076 .079 .078 .111 .564 .717 G .895.094 .072 .077 .077 .079 .077 .077 .080 .074 .069

Example #3

[0075] Table 7 shows the results of incubation of Escherichia colibiofilm on projections 12 in channels 24 while rocking the vessel 20,followed by treatment with antibiotics A (Ticarcillin, Sigma T-5639), B(Carbenicillin, Sigma C-1389), C (Tobramycin, Sigma T-4014), D(Gentamicin sulphate, Sigma G-3632), E (Ampicillin, Sigma A-9518), F(Tazadine, ceftazidine), G (Primaxin, imipenem and cilastatin) and H(Ciprofloxacin) in doubling dilutions. The Escherichia coli was startedwith an inoculum of 2% of overnight growth in fresh TSB. 20 ml wereplaced in the main basin of the vessel 20 (channels 2-12), with 1.5 mlin the channel 1 to provide a sampling of initial colonization on theprojections of the first row. The initial biofilm was colonized for fourhours. The antibiotic was applied to the wells 2-12 of the rows A-H of a96 well plate as follows: well 2: no antibiotic, well 3: 1000 μg/mL,well 4: 500 μg/mL . . . well 12: 2 μg/mL. 250 μL final volume of dilutedantibiotic was applied to the wells. TABLE 7 2 3 4 5 6 7 8 9 10 11 12 A1.9 × 0 0 0 0 0 3.5 × 2.0 × 2.5 × 1.5 × 3.0 × 10⁵ 10² 10² 10² 10⁴ 10⁴ B2.0 × 1.5 × 0 0 0 1.5 × 0 2.0 × 2.0 × 1.2 × 1.1 × 10⁸ 10² 10² 10² 10³10⁵ 10⁷ C 1.0 × 0 0 0 0 0 0 0 2.5 × 1.1 × 1.3 × 10⁷ 10⁴ 10⁶ 10⁷ D 1.5 ×0 0 0 0 0 0 0 1.5 × 1.5 × 2.5 × 10⁷ 10³ 10⁶ 10⁶ E 8.5 × 2.0 × 0 1.1 ×4.0 × 2.0 × 0 1.5 × 8.5 × 6.5 × 5.0 × 10⁷ 10⁵ 10⁵ 10⁵ 10⁶ 10⁵ 10⁵ 10⁷10⁷ F 1.2 × 0 0 0 0 0 0 0 2.0 × 1.5 × 1.5 × 10⁸ 10³ 10⁴ 10⁴ G 1.0 × 0 02.0 × 0 0 0 0 0 0 0 10⁷ 10² H 1.1 × 0 0 0 0 0 0 1.5 × 0 0 2.0 × 10³ 10²10²

[0076] Table 8 shows the optical density readings of turbidity of thesame Escherichia coli biofilm, treated in the same manner as the samplesof Table 7, to show a comparison between the manually counted cfu andthe automated reading. TABLE 8 2 3 4 5 6 7 8 9 10 11 12 A 1.296 .120.118 .121 1.064 1.099 .661 1.097 1.130 1.244 1.250 B 1.328 1.181 .6691.247 .876 .769 .653 1.114 1.177 1.192 1.297 C 1.289 .121 .122 .121 .122.122 .119 .666 .647 .829 1.299 D 1.260 .124 .121 .123 .121 .132 .123.672 1.184 1.296 1.294 E 1.345 1.053 .124 1.046 1.114 1.544 .797 1.3341.303 1.573 1.332 F 1.341 .121 .123 1.158 .121 .125 .120 .123 1.1521.249 1.268 G 1.313 .124 1.142 1.215 1.235 1.284 .773 1.180 1.117 1.274.637 H 1.317 .122 .126 .118 .119 .123 .786 1.011 .969 .940 1.233

[0077] Table 9 shows the optical density readings of turbidity ofEscherichia coli planktonic bacteria incubated and treated withantimicrobial reagent under the same conditions as for the results shownin Table 7. This table clearly shows how the assay of planktonicbacteria gives a lower count in many instances than the assay of sessilebacteria incubated and treated under the same conditions. TABLE 9 2 3 45 6 7 8 9 10 11 12 A 1.080 .119 .132 .137 .135 .137 .138 .154 .143 .159.273 B 1.212 .120 .135 .134 .134 .132 .137 .136 .185 .252 .652 C .828.122 .132 .136 .136 .133 .141 .133 .588 .601 .873 D 1.040 .125 .133 .138.135 .141 .141 .136 .334 .837 .936 E 1.040 .124 .129 .133 .138 .142 .139.254 .231 .629 1.425 F 1.098 .123 .129 .134 .137 .128 .137 .137 .140.147 .152 G .560 .206 .190 .177 .161 .146 .148 .141 .135 .139 .153 H1.147 .131 .136 .136 .140 .132 .139 .136 .138 .133 .138

[0078] The concentration (MBEC) of antimicrobial reagent at which thesurvival of bacteria falls to zero may be assessed readily from theassay. Likewise, the MIC may also be determined from the assay.

[0079] The inventors have found that in some instances a biofilm willnot form without the inclusion of host components in the biofilm. Hostcomponents may therefore be added to the growth medium in the vesselduring incubation of the bacteria or other biofilm forming organisms toform the biofilm. Host components that may be added include serumprotein and cells from a host organism. For the testing of the effect ofdifferent host cells and components, the ends 25 of the channels 24 maybe sealed by walls to prevent growth medium in one channel from flowinginto another, thus isolating the biofilm growth in each channel fromother channels.

[0080] The device thus described may also be used to test coatings usedto inhibit biofilm growth and to test coatings which may enhance biofilmformation. In an initial step, the projections 12 may be coated with acoating to be tested, and then the biofilm grown on the projections. Thebiofilm may then be assayed, or treated with antimicrobial reagent andthen assayed. The assay may be in situ or after dislodging of thebiofilm. Different coatings may be tested on different rows of pegs.Enhanced biofilm formation may be used to create large viable biofilmsfor bio-fermentation under varying growth conditions at different sites.In this instance, the assay step is not required, except to assay thefermentation product, if desired, and the biofilm is incubated in thewells of, for example, a 96 well plate as a micro-reactor vessel. Ingeneral, the biofilm incubation system disclosed may be used wheneverrapid and uniform growth of biofilm is desired at plural sites.

[0081] While the preferred technique is to reverse flow of the liquidgrowth medium, the array could have a unidirectional flow of liquid,with recircling of fluid from one end of each channel to the other endof the same channel, but this complicates the arrangement.

[0082] The method of the invention may be used to screen potentialantimicrobial candidates for efficacy. After incubation of the biofilmeach biofilm adherent site may be treated with a different antimicrobialreagent or combination of antimicrobial reagents. For example, thedifferent reagents may be modifications of a single reagent, such asmight be produced in recombinatorial development of antibiotics. In aplate with 340 pins, 340 different combinations of antimicrobialreagents may be tested.

[0083] A person skilled in the art could make immaterial modificationsto the invention described in this patent document without departingfilm the essence of the invention that is intended to be covered by thescope of the claims that follow.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of growing abiofilm, the method comprising the steps of: incubating biofilm formingorganisms on plural biofilm adherent sites while providing a flow ofliquid growth medium across the plural biofilm adherent sites, thedirection of the flow of liquid being repeatedly changed, to therebycreate a biofilm at the plural biofilm adherent sites.
 2. The method ofclaim 1 further comprising the step of assaying the biofilm at theplural biofilm adherent sites to analyze a characteristic of thebiofilm.
 3. The method of claim 2 in which the characteristic of thebiofilm is the sensitivity of the biofilm to antimicrobial reagent andthe method further comprises the step of, before assaying the biofilm,treating the biofilm adherent sites with antimicrobial reagent.
 4. Themethod of claim 3 further comprising: after treating the biofilmadherent sites with antimicrobial reagent, dislodging the biofilm fromthe biofilm adherent sites before performing the assay.
 5. The method ofclaim 4 further including, after dislodging the biofilm from the biofilmadherent sites, further incubating the biofilm.
 6. The method of claim 4in which dislodging the biofilm from the biofilm adherent sites includesdislodging the biofilm from each biofilm adherent site into a separatewell of a well-plate.
 7. The method of claim 3 in which the biofilmadherent sites are formed in rows, and treating the biofilm adherentsites with an antimicrobial reagent includes: treating each row ofbiofilm adherent sites with a different antimicrobial reagent.
 8. Themethod of claim 7 further including treating each biofilm adherent sitein a row with a different concentration of antimicrobial reagent.
 9. Themethod of claim 3, 7 or 8 in which flow direction of the liquid growthmedium is repeatedly reversed.
 10. The method of claim 9 in which theliquid growth medium flows in channels of a vessel, and the direction offlow of the liquid growth medium is reversed by rocking of the vessel.11. The method of claim 10 in which the biofilm adherent sites areprojections from a lid and incubating the biofilm includes: suspendingthe projections in liquid growth medium in the channels while rockingthe vessel so as to provide shear forces on the biofilm during growth ofthe biofilm.
 12. The method of claim 2 further including treating eachof the biofilm adherent sites with a different concentration ofantimicrobial reagent.
 13. The method of claim 12 further including:before incubating biofilm forming organisms on plural biofilm adherentsites, applying a biofilm resistant coating to the plural biofilmaderent sites.
 14. The method of claim 13 further including applyingdifferent coatings to different biofilm adherent sites.
 15. The methodof claim 1 further including: incubating the biofilm forming organismsin the presence of host material in the liquid growth medium.
 16. Themethod of claim 1 further including the step of, after initiallyincubating the biofilm forming organisms on plural biofilm adherentsites to create a biofilm, subsequently incubating the biofilm formingorganisms in separate micro-reactors.
 17. The method of claim 3 in whichdifferent biofilm adherent sites are treated with differentantimicrobial reagents.
 18. Apparatus for analyzing biofilms, theapparatus comprising: a vessel including at least one channel for flowof liquid growth medium; plural biofilm adherent sites having a supportfor supporting the plural biofilm adherent sites within the channel; andmeans to flow liquid growth medium along each channel in differentdirections across the plural biofilm adherent sites.
 19. The apparatusof claim 18 in which: the plural biofilm adherent sites are formed inplural rows, with more than one biofilm adherent site in each row; thevessel includes plural channels having substantially the same shape. 20.The apparatus of claim 19 in which the biofilm adherent sites havesubstantially the same shape.
 21. The apparatus of claim 20 in which thesupport for the plural biofilm adherent sites forms a lid for thevessel.
 22. The apparatus of claims 18, 19 or 20 in which the means toflow liquid growth medium across the plural biofilm adherent sites is atilt table.
 23. A method of growing a biofilm, the method comprising thesteps of: incubating biofilm forming organisms on plural biofilmadherent sites arranged in plural rows, with plural biofilm adherentsites in each row, while providing a flow of liquid growth medium acrosseach of the plural biofilm adherent sites, to thereby create a biofilmat each of the plural biofilm adherent sites.
 24. The method of claim 23further comprising the step of assaying the biofilm at the pluralbiofilm adherent sites to analyze a characteristic of the biofilm. 25.The method of claim 24 in which providing a flow of liquid growth mediumincludes flowing liquid growth medium backwards and forwards alonguniform channels of a vessel to create a uniform biofilm at each biofilmadherent site.
 26. The method of claim 24 in which the characteristic ofthe biofilm is the sensitivity of the biofilm to antimicrobial reagentand the method further comprises the step of, before assaying thebiofilm, treating the biofilm adherent sites with antimicrobial reagent.27. The method of claim 26 further comprising: after treating thebiofilm adherent sites with antimicrobial reagent, dislodging thebiofilm from the biofilm adherent sites before assaying the biofilm. 28.The method of claim 27 further including, after dislodging the biofilm,further incubating the biofilm.
 29. The method of claim 27 in whichdislodging the biofilm from the biofilm adherent sites includes:dislodging the biofilm from each biofilm adherent site into a separatewell of a well plate.
 30. The method of claim 29 in which treating thebiofilm adherent sites with an antimicrobial reagent includes treatingeach row of biofilm adherent sites with a different antimicrobialreagent.
 31. The method of claim 30 further including: treating eachbiofilm adherent site in a row with a different concentration ofantimicrobial reagent.
 32. The method of claim 23 in which the biofilmadherent sites are projections from a lid and incubating the biofilmincludes: suspending the projections in liquid growth medium in uniformchannels of a vessel while rocking the vessel so as to provide uniformshear forces on the projections during growth of the biofilm.
 33. Themethod of claim 24 in which assaying the biofilm comprises applying avital dye to the biofilm and automatically measuring the intensity oflight given off by the dyed biofilm.
 34. The method of claim 23 furtherincluding the step of, after initially incubating the biofilm formingorganisms on plural biofilm adherent sites to create a biofilm,subsequently incubating the biofilm forming organisms in separatemicro-reactors.
 35. The method of claim 23 further including: beforeincubating biofilm forming organisms on the plural biofilm adherentsites, applying a biofilm resistant coating to the plural biofilmadherent sites.
 36. The method of claim 35 further including applyingdifferent coatings to different biofilm adherent sites.
 37. The methodof claim 23 further including incubating the biofilm forming organismsin the presence of host material in the liquid growth medium.
 38. Themethod of claim 26 in which different biofilm adherent sites are treatedwith different antimicrobial reagents.
 39. Apparatus for growingbiofilms, the apparatus comprising: a vessel including plural channelsfor flow of liquid growth medium; plural biofilm adherent sites arrangedin plural rows and having a support for supporting the plural biofilmadherent sites within the channels; and means to avoid contamination ofthe inside of the vessel during incubation.
 40. The apparatus of claim39 in which each channel has substantially the same shape.
 41. Theapparatus of claim 40 in which each biofilm adherent site hassubstantially the same shape.
 42. The apparatus of claim 41 furtherincluding: means to flow liquid growth medium along each channel acrossplural biofilm adherent sites supported within the channel.
 43. Theapparatus of claims 41 or 42 in which: the support for the pluralbiofilm adherent sites forms a lid for the vessel.
 44. The apparatus ofclaim 43 in which the plural biofilm adherent sites are projections fromthe lid.
 45. The apparatus of claim 42 in which the means to flow liquidgrowth medium across the plural biofilm adherent sites is a tilt table.